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Evaluation of in Vitro Spermatogenesis System Effectiveness to Study Genes Behavior: Monitoring the Expression of the Testis 6SHFL¿F 7VJD *HQHDVD0RGHO

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Evaluation of in Vitro Spermatogenesis System Effectiveness to Study Genes Behavior: Monitoring the Expression of the Testis 6SHFL¿F 7VJD *HQHDVD0RGHO In vitro*HQHUDWLRQRI*HUP&HOOV7VJDDVD0RGHO Original Article Evaluation of in vitro Spermatogenesis System Effectiveness to Study Genes Behavior: Monitoring the Expression of the Testis 6SHFL¿F 7VJD *HQHDVD0RGHO Mohammad Miryounesi MD PhD1,2, Karim Nayernia PhD3, Maryam Beigom Mobasheri MSc2,4, Mahdi Dianatpour PhD5, Richard Oko PhD6, Shahram Savad MD PhD7, Mohammad Hossein Modarressi MD PhD2 Abstract Background: In vitro generation of germ cells introduces a novel approach to male infertility and provides an effective system in gene tracking studies, however many aspects of this process have remained unclear. We aimed to promote mouse embryonic stem cells (P(6&) differentiation into germ cells and evaluate its effectiveness with tracking the expression of the Tsga10 during this process. Methods: P(6&V were differentiated into germ cells in the presence of Retinoic Acid. Based on developmental schedule of the postnatal testis, samples were taken on the 7th, 12th, and 25th days of the culture and were subjected to expression analysis of a panel of germ cell VSHFL¿F genes. ([SUHVVLRQ of Tsga10 in RNA and protein levels was then analyzed. Results: Transition from mitosis to meiosis occurred between 7th and 12th days of P(6& culture and post-meiotic gene expression did not occur until the 25th day of the culture. Results showed low level of Tsga10expression in undifferentiated stem cells. During transition from meiotic to post-meiotic phase, Tsga10 expression increased in 6.6 folds. This ¿QGLQJ is in concordance with in vivo changes during transition from pre-pubertal to pubertal stage. Localization of processed and unprocessed forms of the related protein was similar to those in vivo as well. Conclusions: ([SUHVVLRQ pattern of Tsga10, as a gene with critical function in spermatogenesis, is similar during in vitro and in vivo germ cell generation. The results suggest that in vitro derived germ cells could be a trusted model to study genes behavior during spermatogen- esis. Keywords: Tsga10; embryonic stem cells; differentiation; in vitro germ cells generation Cite this article as: Miryounesi M, Nayernia K, Mobasheri MB, Dianatpour M, Oko R, Savad S, Modarressi MH. Evaluation of in vitro Spermatogenesis System Effectiveness to Study Genes Behavior: Monitoring the Expression of the Testis Specific 10 (Tsga10) Gene as a Model. Arch Iran Med. 2014; 17(10): 692 – 697. Introduction Consequently we tried to create an in vitro spermatogenesis sys- tem and monitor a testis VSHFL¿F gene, Tsga10, in the process of bout 15% of couples have fertility problems and male fac- stem cells differentiation, and compare it with in vivo system. This tor infertility accounts for half of the cases.1 Numerous gene has been studied in our team in several research projects and A pathologic conditions are involved in infertility prob- adequate information about its in vivo characteristics has been ob- lems.2 To elucidate the causes of male infertility, it is important to tained. know about the mechanisms of germ cell VSHFL¿FDWLRQ, develop- The Tsga10 is mainly expressed in the testis tissue. It has been ment and differentiation. Due to lack of a robust in vitro culture shown that Tsga10 has a critical role in spermatogenesis and its system for differentiation of germ line progenitor cells into mature protein contributes to the ¿EURXV sheath of sperm tail.5 In addition gametes, enough knowledge in this area is not available. Tsga10 is expressed in actively dividing cells and fetal tissues.6 Recent studies have shown that embryonic stem cells (ESC) can This gene is a member of cancer testis (CT)7,8 genes family. Its give rise to primordial germ cells (PGCs) and sperm-like cells in aberrant expression is reported in acute lymphoblastic leukemia vitro.3 This ability provides an in vitro model to analyze the biol- and some primary cancers.9 Tsga10 mRNA is translated into a ogy of germ cells development and new therapeutic approaches in 65-kilodaltone protein. During spermatogenesis, it is processed to reproductive medicine.4 a 27-kilodalton N-terminal segment. This portion is localized to the ¿EURXV sheath of mature spermatozoa.10 $XWKRUV¶DI¿OLDWLRQV 1Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2Department of Medical Genetics, School of In this study, for the ¿UVW time the alterations in Tsga10 expres- Medicine, Tehran university of Medical Sciences, Tehran, Iran. 3GENEOCELL, sion during in vitro differentiation of embryonic stem cells to 4 Advanced Molecular and Cellular Technologies, Montreal, Canada. Cancer Re- germ cells was analyzed. search Center, Tehran University of Medical Sciences, Tehran, Iran. 5Department of Medical Genetics, School of Medicine, Shiraz university of Medical Sciences, To FRQ¿UP the different developmental stages of the differenti- Shiraz, Iran. 6Departments of Anatomy and Cell Biology, Queen’s University, 9th ated cells, the expression panel of 6 stage VSHFL¿F genes, Stra8, 7 Floor, Botterell Hall, Kingston, Ontario, Canada K7L 3N6. Kamali Hospital, Sycp3, Dazl, Prm1, Spata19, and Plcz in the development process Alborz University of Medical Sciences, Karaj, Iran. &RUUHVSRQGLQJ DXWKRU DQG UHSULQWV Mohammad Hossein Modarressi MD was studied. PhD, Department of Medical Genetics, School of Medicine, Tehran University Retinoic Acid (RA) is now FRQ¿UPHG to be a key signaling mol- of Medical Sciences, Tehran, Iran. Telefax: +98-218-895-3005, ecule in meiosis induction.11 RA induces a VLJQL¿FDQW increase in E-mail: [email protected] Accepted for publication: 16 July 2014 the expression of Stra8 (a pre-meiotic gene) and Sycp3 (a meiotic 692 Archives of Iranian Medicine, Volume 17, Number 10, October 2014 00LU\RXQHVL.1D\HUQLD0%0REDVKHULHWDO gene) in cultures of human fetal testis.12 According to gene knock- for two weeks. Resulting colonies were cultured for 25 days on out studies, Stra8 is believed to be the gatekeeper of the mitotic/ gelatin with RA treatment (¿QDO concentration 10-8 mol). meiotic switch.13 Dazl is a testis-VSHFL¿F gene with a critical role in spermatogenesis VSHFL¿FDOO\ in meiosis initiation.14,15 Sycp3 is a RNA extraction, cDNA synthesis and RT-PCR meiotic VSHFL¿F gene responsible for the formation of the synap- Total RNA was extracted using TRIzol reagent (Invitrogen) ac- tonemal complex. This complex has an important role in synapse cording to the manufacturer’s protocol. RNA concentration was formation, recombination and segregation of chromosomes dur- measured with Nano Drop 1000 spectrophotometer (Thermo ing meiosis.16 Sycp3 is positively regulated by Dazl through bind- Fisher 6FLHQWL¿F). The extracted RNA (2 μg) was used for cDNA ing to 3ƍUTR of its transcript. Protamine is coded by Prm1 which synthesis using M-MuLV reverse transcriptase (Fermentase) with is a post-meiotic gene with VSHFL¿F expression in haploid round random hexamer and oligo dT primer together. RT-PCR was per- spermatids. During development of mature spermatozoa histones formed using VSHFL¿F primer for EGFP, Oct4, Stra8, Sycp3, Dazl, are replaced by protamines.17,18 Spata19 is a testis VSHFL¿F gene Prm1, Plcz, and Spata19 genes (Table 1). that is expressed VSHFL¿FDOO\ in round spermatids.19 Finally, Plcz is the physiological agent of oocyte activationand its protein causes Real time RT-PCR Ca2+ osciliation in oocyte similar to those seen in fertilization. It Real time RT-PCR was carried out using VSHFL¿F primer for was shown that Plcz mRNA is ¿UVW detectable in spermatids.20 Tsga10 gene with StepOne Real time PCR system (Applied Biosystems). Typically, a 20 μL reaction contained 10μL 2 × Materials and Methods SYBR® Green PCR Master Mix (Applied Biosystems), 1 μL (¿- nal 500nM) forward and reverse primer, 1 μL of cDNA and 8 μL Cell culture distilled water. Expression analysis of Tsga10 gene and a house- Mouse embryonic stem cell line C57BL6 with normal male keeping gene, Tbp (Tata Binding Protein) were performed using (XY) karyotype (Invitrogen) was cultured in an undifferenti- StepOne Software v2.1 (Applied Biosystems). ated state on a feeder layer of mitomycin-C inactivated mouse embryonic ¿EUREODVW.21,22 Culture medium composed of Knock- Western blot analysis out Dulbecco’s PRGL¿HG Eagle’s medium (Knockout DMEM, Equal amounts of cell lysate proteins of selected samples (adult GIBCO-BRL) supplemented with 12.5% (v/v) ES TXDOL¿HG FBS testis and differentiated ES) were separated using 10% SDS- (GIBCO-BRL), 2mmol L-Glutamine (GIBCO-BRL), 1X nones- PAGE and transferred to a Polyvinylidene ÀXRULGH (PVDF) mem- sential amino acids (NEAA; GIBCO-BRL), 50 μgml-1 penicillin brane (Milipore). The membrane free binding sites were blocked and streptomycin, as well as 50 μmol ȕ-mercaptoethanol and 10³ by treating the membrane with 5% (wt/ vol) skim milk for one unit ml-1 LIF (Milipore). hour at room temperature. After overnight incubation at 4°C with primary Tsga10 goat polyclonal antibody (Santa Cruz), blots were &RQVWUXFWLRQ RI JHUP FHOO VSHFL¿F UHSRUWHU JHQH DQG UHFRPELQDQW washed 3 times with PBST. After incubation with the appropri- mESCs ate horseradish peroxidase-labeled secondary antibody (anti goat, According to our previous published data, a segment of Stra8 Santa Cruz), blots were washed again as mentioned previously. gene promoter (-1400/+7) was DPSOL¿HG from genomic DNA Bands were detected using enhanced chemiluminescence (ECL). and inserted in the SacI/HindIII site of PRGL¿HG pEGFP-1 vector where neomycin-resistance cassette was replaced with puromy- Immunohistochemistry of TSGA10 in seminiferous tubules cin-resistance gene (Figure 2d).23 ES cells were trypsinized and Sections from testes (5-ȝP) ¿[HG in Bouin’s and embedded in paraf- approximately 7 × 106 cells were prepared for performing elec- ¿Q, were GHSDUDI¿QL]HG in xylene and hydrated through a graded se- troporation. Linearized vector (35 μg) was electroporated into ries of ethanol solutions. During hydration the sections were treated to ES cells.
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